In Vitro Stimulation and Visualization of Macrophage Extracellular Traps

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1. Polarization of HMDM

1. Under sterile conditions, prepare the M1 priming media by adding interferon γ (IFNγ; 20 ng/mL) and lipopolysaccharide (LPS; 1 μg/mL) to the complete RPMI-1640 culture media. Prepare the M2 priming media by adding interleukin 4 (IL-4; 20 ng/mL) to the complete RPMI-1640 culture media.
2. Under sterile conditions, aspirate media from the tissue culture plate wells that contain the Human monocyte-derived macrophages, HMDM, which have been seeded and cultured as described in section 1.
3. Carefully wash the wells containing the cells 3x with sterile PBS (pre-warmed to 37 °C), using 1 mL aliquots of PBS.
4. Add 1 mL of either the M1 or M2 priming media to each well containing the HMDM (whichever is appropriate for the experiment).
5. Incubate the cells for 48 h at 37 °C in the presence of 5% CO₂ in a cell incubator.

2. Stimulation of HMDM to induce MET Release

1. Under sterile conditions, prepare the culture media containing different stimulators of macrophage extracellular traps MET release (whichever is appropriate for the experiment) to the complete RPMI-1640 media: PMA (25 nM), human recombinant TNFα (25 ng/mL), or human recombinant IL-8 (50 ng/mL).
2. For experiments with hypochlorous acid, HOCl stimulation, prepare HOCl (200 μM) in HBSS (pre-warmed to 37 °C), immediately before the addition to the cells. Ensure that the HOCl is not prepared in complete cell media.
   NOTE: The concentration of the stock solution of HOCl is quantified by measuring the UV absorbance of the solution at 292 nm and pH = 11⁶ and using an extinction coefficient of 350 M^-1cm^-1.
3. After the polarization treatment described in section 2, aspirate the cell media from each well and carefully wash the cells 3x with 1 mL aliquots of either: sterile PBS (for PMA, TNFα and IL-8) or HBSS (for HOCl), which have been pre-warmed to 37 °C.
4. For experiments with PMA, TNFα, or IL-8: add 1 mL of the complete media containing PMA, TNFα, or IL-8 after removing the PBS in the final washing step.
5. For experiments with TNFα, incubate the cells for 6 h at 37 °C in the presence of 5% CO2 in a cell incubator. For experiments with PMA and IL-8, incubate the cells for 24 h at 37 °C in the presence of 5% CO₂ in a cell incubator.
6. For experiments with HOCl, add 1 mL of HOCl in HBSS after removing the HBSS in the final washing step. Then, incubate the cells for 15 min at 37 °C in the presence of 5% CO₂ in a cell incubator.
   1. Carefully aspirate the cell supernatant and wash the cells 3x with 1 mL aliquots of HBSS as described in step 2.3.
   2. After removing the HBSS from the final wash step, add 1 mL of complete RPMI-1640 culture media. Then, incubate the cells for 24 h at 37 °C in the presence of 5% CO₂ in a cell incubator.

3. Visualization of MET in Live Cell Culture

1. Prepare SYTOX green dye in HBSS at a concentration of 40 μM.
2. At the end of treatments described in section 2 to induce MET release, directly add 25 μL of 40 μM of the dye to each well containing HMDM.
3. Incubate cells at room temperature (RT) for 5 minutes in the dark.
4. Place the HMDM in tissue culture wells on the microscope stage of an inverted fluorescent microscope for imaging.
5. Microscope procedures
   1. Turn on a broad-spectrum fluorescent light source, brightfield light source, and inverted microscope installed with a high-resolution color digital camera (see Table of Materials).
   2. Rotate the filter wheel to the "number 2" position for green fluorescence (excitation = 504 nm, emission = 523 nm) for imaging of the green stained samples contained within the tissue culture wells.
   3. Using the 5x objective, focus the image with the coarse focus, then the fine focus knobs on the microscope, until the image appears sharp, clear, and focused when viewed through the microscope eyepiece.
   4. Switch the microscope to the camera mode.
   5. Start the associated software.
   6. Select the Capture tab on the software.
   7. Click the Play button to preview the image and adjust the fine focus knob on the microscope until the image appears sharp, clear, and focused in the software preview window.
   8. Click the Capture button.
      NOTE: The captured image will automatically be displayed in the accompanying software.
   9. Within the software, click File | Save as the required image file type.
   10. On the microscope, rotate the filter wheel to the "number 5" position for brightfield imaging and repeat steps 3.5.2–3.5.9 to obtain the corresponding brightfield image.
   11. Repeat the steps 3.5.2–3.5.10 as necessary for subsequent image acquisition.

자료

Name	Company	Catalog Number	Comments
120Q broad spectrum fluorescent light source	EXFO Photonic Solutions, Toronto, Canada	x-cite series
Corning CellBIND Multiple Well Plate (12 wells)	Sigma-Aldrich	CLS3336	For cell culture
Hypochlorous acid (HOCl)	Sigma-Aldrich	320331	For MET stimulation
Interferon gamma	Thermo-Fisher	PMC4031	For M1 priming
Interleukin 4	Integrated Sciences	rhil-4	For M2 priming
Interleukin 8	Miltenyl Biotec	130-093-943	For MET stimulation
L-Glutamine	Sigma-Aldrich	59202C	Added to culture media
Olympus IX71 inverted microscope	Olympus, Tokyo, Japan
Phorbol 12- myristate 13-acetate (PMA)	Sigma-Aldrich	P8139	For MET stimulation
Phosphate buffered saline (PBS)	Sigma-Aldrich	D5652	For washing steps
RPMI-1640 media	Sigma-Aldrich	R8758	For cell culture
SYTOX green	Life Technologies	S7020	For MET visulaization
TH4-200 brightfield light source	Olympus, Tokyo, Japan		x-cite series
Tumor necrosis factor alpha	Lonza	300-01A-50	For MET stimulation